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PubMed1972

Immunofluorescence staining of group B coxsackieviruses.

Studies were conducted on the sensitivity and specificity of indirect fluorescent-antibody (FA) staining for identification of group B coxsackieviruses. Antisera produced in four different species (monkeys, rabbits, horses, hamsters) and immune ascitic fluids prepared in mice were compared for suitability in FA staining. The horse antisera showed high titers of nonspecific staining, and the rabbit antisera showed relatively low homologous FA titers. Immune reagents from monkeys, hamsters, and mice were used for homologous and heterologous testing against cell cultures infected with the various group B coxsackieviruses. Antisera or immune ascitic fluids produced in these three species showed some heterotypic and nonspecific staining at low dilutions, with the monkey antisera showing the highest heterotypic titers. However, the immune reagents could be diluted to a point where they gave no heterotypic reactivity, but still showed characteristic homotypic staining. Heterotypic staining appeared as diffuse, low-level staining of the cells, whereas homotypic staining revealed characteristic, brightly staining aggregates of viral antigen in the cytoplasm of the infected cells. By using hamster immune sera, appropriately diluted to eliminate heterotypic staining and yet give strong homotypic staining, it was possible to identify correctly 79 (93%) of 85 field strains of group B coxsackieviruses at the first passage level in BS-C-1 cells; the remainder of the strains were identified after two passages in BS-CS-1 cells. No incorrect identifications were made. A limited number of field strains of group B coxsackieviruses were passed into rhesus monkey kidney and human fetal diploid kidney cells, and these were all correctly identified by FA staining, even the strains which failed to produce a cytopathic effect in the human fetal diploid kidney cells. Two human heart and brain tissues from which coxsackievirus type B4 had been isolated failed to show homotypic FA staining in excess of nonspecific or heterotypic staining.

Animals↗

PubMed2002

The effectiveness of two different battery-powered toothbrushes on whitening through removal of stain.

Whitening dentifrices have recently become popular, achieving a whitening benefit by surface chemical action and/or abrasion that serve to remove extrinsic stains and/or prevent extrinsic stain buildup. Some powered toothbrushes have also been demonstrated to have whitening/stain-removal efficacy when used with standard dentifrice products. The objective of this study was to evaluate the effectiveness of an experimental prototype powered toothbrush (Crest SpinBrush Pro) and a commercially available powered toothbrush (Crest SpinBrush) on dental whitening through the removal of extrinsic stain. This study was a randomized, controlled, examiner-blind, parallel-group design, which examined extrinsic stain removal over four weeks of brushing by 70 subjects. Following a three-week period of stain induction using rinses of chlorhexidine and tea, subjects were randomized to use one of the two toothbrushes. Tooth stain was scored using the Lobene stain index at baseline, and after two and four weeks of toothbrush use. Prior to statistical analysis, the stain scores were averaged on a per-subject basis. The stain reductions (baseline minus post-treatment) in average scores were calculated and analyzed using an analysis of covariance, with baseline average score as the covariate. The experimental prototype powered toothbrush (Crest SpinBrush Pro) delivered statistically significant (p < 0.001) adjusted (via analysis of covariance) mean reductions from baseline in Lobene composite stain scores of 1.90 and 2.11 after 2 weeks and 4 weeks of use, respectively. These results represent 56-62% reductions in extrinsic stain compared to baseline. The commercially available powered toothbrush (Crest SpinBrush) also delivered statistically significant (p < 0.001) adjusted (via analysis of covariance) mean reductions from baseline in Lobene composite stain scores, with magnitudes of 1.95 and 2.22 after two and four weeks of use, respectively. These results represent 60-68% reductions in extrinsic stain compared to baseline. In conclusion, both the experimental prototype powered toothbrush and the commercially available powered toothbrush were effective in removing extrinsic tooth stain.

Adult↗

PubMed1998

Stain removal efficacy: an in vitro evaluation using quantitative image analysis.

OBJECTIVE: This study developed a computer image analysis technique as a quantitative means to measure changes in dental stain after brushing with various dentifrices. METHOD AND MATERIALS: Enamel specimens with naturally occurring mature stain were cut from bovine incisors. The specimens were subjected to in vitro toothbrushing with one of the four tested groups, consisting of two dentifrices that make claims of stain removal (Aquafresh Whitening and Rembrandt Sensitive), a regular dentifrice, (Aquafresh Triple Protection), and water. Digital images of stain specimens were recorded under standardized lighting conditions and analyzed with an image analysis software. The area-intensity stain determinant, which accounted for the reflected intensity and the corresponding areas of stain, was computed. Stain removal efficacy was calculated based on the difference in area-intensity stain determinant before and after brushing. RESULTS: Brushing with any of the tested dentifrices removed more stain than did brushing with water alone. The finding that brushing with a regular dentifrice resulted in higher stain removal efficacy than brushing with water seems to indicate a role for abrasivity. Aquafresh Whitening had a higher stain removal efficacy than did Rembrandt for the removal of mature calcified stain used in this study. However, there were certain stains that none of the dentifrices removed. CONCLUSION: Computer image analysis provides an objective and quantitative measurement to distinguish in vitro stain removal efficacy of dentifrices.

Analysis of Variance↗

PubMed1996

Laboratory implementation of a rapid three-stain technique for detection of microorganisms from lower respiratory specimens.

A rapid, cost-effective method for the evaluation of lower respiratory specimen has become increasingly important in the diagnosis of pulmonary diseases in immunocompromised patients. In the past, the technically demanding, time-consuming, and expensive Gomori-methenamine-silver (GMS) stain was the principal means for the evaluation of these specimens. In this study, we compared the GMS stain with a new rapid, three-stain protocol for the evaluation of lower respiratory specimens. Lower respiratory specimens were obtained by bronchoalveolar lavage (BAL). Conventional Wright/Giemsa and Gram stains were utilized, as well as a contemporary strain, calcofluor white (CW). A cell count was performed on the BAL specimens, and cytospins were stained by the three stains. The calcofluor white-stained slides were examined with an epi-fluorescent microscope, whereas the other stains were evaluated with a conventional light microscope. Gomorimethenamine-silver (GMS), acid-fast bacillus (AFB), and Papanicolaou (PAP) stains were performed as controls. Thirty-two BAL procedures were performed in 20 (63%) male patients and 12 (37%) female patients. The clinical diagnosis was pneumonia in 31% of the patients, malignant hematologic disease in 28%, acute respiratory distress syndrome (ARDS) in 9%, and acquired immunodeficiency syndrome (AIDS) in 28%. Of these specimens, 78% were adequate for interpretation and 22% were inadequate. Bacteria were found in 50% (16/32) of all BALs, fungi were found in 9% (3/32), and Pneumocystis carinii was found in 9% (3/32). Gram-positive bacteria were most frequently found in patients with pneumonia (80%, 4/5), whereas P. carinii was identified in patients with AIDS. There were no false-positive results. One CW stain was equivocal for P. carinii due to high fluorescent background. Laboratory implementation of the rapid, three-staining technique was accomplished without difficulty in microbiology and hematology laboratory sections. Specimen evaluation with the rapid staining protocol was technically easy to perform; however, experience in ultraviolet fluorescent microscopy was crucial for interpretation of CW stain. All results were available in 2 hr, cost was reduced by 30%, and the assays were available 7 days/week. Further studies are ongoing to substantiate the sensitivity, specificity, and predictive value of this technique, as well as clinical guidelines for its optimal utilization.

Adult↗

PubMed1993

Intercellular adhesion molecule-1 (ICAM-1) staining of reactive and neoplastic follicles. ICAM-1 expression of neoplastic follicle differs from that of reactive germinal center and is independent of follicular dendritic cells.

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) is expressed on follicular dendritic cells (FDC), and the ICAM-1/LFA-1 pathway is essential for affinity selection of activated B-cells in germinal centers (GC). The expression of ICAM-1 has been studied by immunostaining methods in GC, but not in neoplastic follicles (NF). METHODS: The authors studied the expression of ICAM-1 by the avidin-biotin-peroxidase complex (ABC) method with frozen sections in GC of reactive nodes (n = 22) and NF of follicular lymphomas (n = 19), in comparison with FDC staining. RESULTS: GC stained uniformly for ICAM-1, with staining intensity varying little from node to node, and follicular borders were well demarcated from the surroundings. Endothelial cells within GC did not stain whereas those outside stained strongly. In contrast, NF stained variably from tumor to tumor, and in any given tumor. Characteristically, follicular borders were not demarcated, and stained areas were difficult to define due to the lack of demarcation, with the exception of large cell type with diffuse areas (n = 2). Endothelium of intrafollicular vessels stained prominently (12 of 19), and appeared to contribute to the staining intensity of the follicles. FDC staining revealed a meshwork pattern in GC which was similar to that of ICAM-1. The FDC mesh-work pattern was present in 15 tumors. The pattern was remarkably uniform with sharply defined borders, which contrasted starkly to the variable staining and lack of follicular borders in the ICAM-1 stain of the same tumors. CONCLUSIONS: The staining pattern of follicles for ICAM-1 was similar to that for FDC in reactive nodes, but distinct from the latter in follicular lymphomas. An appropriate expression of ICAM-1 appears to be essential for normal GC, as the alteration of the expression coincides with the malignant transformation of GC. The altered expression of ICAM-1 may be useful in distinguishing NF from reactive GC.

Adolescent↗

PubMed1996

Applications of SYPRO orange and SYPRO red protein gel stains.

We have further characterized the sensitivity and specificity of SYPRO Orange protein gel stain and SYPRO Red protein gel stain with native and 2-dimensional polyacrylamide gels and for staining gels prior to Western blot analysis. We found that nucleic acids are not stained by the SYPRO protein gel stains, in contrast to results obtained with commonly used silver staining techniques. Bacterial lipopolysaccharides also stain very weakly with SYPRO dyes in comparison to silver staining. Thus, gels containing whole cell lysates from either bacterial or mammalian cells can be analyzed more easily with the SYPRO stains than by silver staining. In this paper, we demonstrate that SYPRO stains can be used to monitor protein induction in bacterial overproducing strains and are effective stains for 2-dimensional gels. In addition, we developed protocols to detect proteins with these dyes in native gels. Finally, we found that staining proteins in transfer buffer prior to Western blotting does not affect the sensitivity of subsequent immunodetection.

Blotting, Western↗

PubMed1993

Comparison of triphenyltetrazolium chloride (TTC) staining versus detection of fibronectin in experimental myocardial infarction.

Staining with triphenyltetrazolium chloride (TTC), although controversial, has frequently been used for the delineation of myocardial infarction. This study was performed further to explore the reliability of the TTC method. In 24-h experiments pigs were subjected to closed-chest occlusion of the left anterior descending coronary artery for 30, 60 or 90 min followed by reperfusion with or without superoxide dismutase (SOD) as an adjunct. One TTC-stained slice from each heart was stabilized by microwave irradiation, gelatin-embedded, frozen in hexane chilled with dry ice and cryosectioned. Serial sections were stained with antibodies against fibronectin in order to identify irreversibly injured myocytes and with van Gieson histologically to confirm the necrotic tissue. A close correspondence of the infarct size was found between TTC stained slices and anti-fibronectin stained sections. The infarct size in the van Gieson stained sections also showed good correspondence but the area of infarction tended to be larger. In the experimental group subjected to 30 min ischaemia and with SOD as an adjunct, the estimated infarcted area in the TTC stained slices was significantly smaller than the area estimated from the anti-fibronectin stained sections. In sections viewed in the light microscope an inverse pattern of TTC and anti-fibronectin staining was observed. It was confirmed at the light microscopic level that myocytes containing an abundance of TTC deposits lacked fibronectin whereas myocytes stained with antifibronectin in general lacked TTC staining except for a zone approximately 0.5 mm wide which was located at the intersection between damaged and surviving myocytes where small TTC deposits were present. The width of the stained zone did not differ among the experimental groups. Thus, differences in estimated infarct size by the three methods used reflect problems in correctly delineating the border between living and dead myocardium rather than an interference by SOD on TTC staining.

Animals↗

PubMed1982

Post-embedding staining of rat gastric mucous cells with lectins.

The mucous cells of the rat stomach were stained with lectins by two post-embedding staining methods for electron microscopy. The mucous granules of surface mucous cells and foveolar mucous cells were stained weakly by Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of mucous neck cells were stained by concanavalin A-ferritin, Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of pyloric gland cells showed an affinity for wheat germ agglutinin-ferritin and concanavalin A-ferritin, while Ricinus-communis agglutinin-ferritin only slightly stained the granules. The granules of mucous neck cells and pyloric gland cells were also stained by the concanavalin A-horseradish peroxidase-colloidal gold method, but the granules of surface and foveolar mucous cells were not stained by this method. Periodic acid oxidation of the sections before the standard concanavalin A-ferritin procedure enhanced the staining of the granules of mucous neck cells and pyloric gland cells slightly. Reduction of the sections after the periodic acid oxidation weakened the staining. Similar results were obtained using the concanavalin A-horseradish peroxidase-colloidal gold method. Though the staining with Ricinus communis agglutinin-ferritin was inhibited by periodic acid oxidation of the sections before staining, the staining with wheat germ agglutinin-ferritin was not inhibited by the oxidation. It is suggested that the paradoxical staining is closely related to the position of the concanavalin A-binding sugar residues in the carbohydrate chains.

Animals↗

PubMed1988

Victoria blue B--a nuclear stain for cytology. A cytophotometric study.

The aim of this study was to investigate the staining characteristics of Victoria Blue B in alcohol solutions. Cytological specimens (liver and spleen tissue imprints, blood smears) were stained with methanol solutions of commercially available Victoria Blue B-Cl and with pure Victoria Blue B-BF4. The dye concentration, staining time, and protone concentration of the dye solution were varied. The dye solutions were characterized using spectrophotometry and thin-layer chromatography. Cytophotometry and image analysis were used to quantitate the staining pattern of cell nuclei. Feulgen-stained slides were used as controls. Victoria Blue B-BF4 gave excellent nuclear staining exhibiting a quantitative dye-substrate relationship, whereas commercial dyes resulted in lower staining intensity and less distinct nuclear texture. Dye concentration and staining time were, over wide ranges, not of critical importance for the quality of the staining. Under certain staining conditions, only cell nuclei were stained, with the background remaining completely unstained. We presume that, in alcohol solutions, Victoria Blue dye binds as a neutral dye molecule in conjunction with its anion. Victoria Blue B-BF4 staining provides a simple and reproducible staining technique for cytology which is suitable for use in automated cell-pattern recognition.

Animals↗

PubMed2001

Immunohistochemical double-staining of renal allograft tissue: critical assessment of three different protocols.

Quantitative analyses of renal allograft tissue using immunohistochemical double-staining could be a useful tool to extend the existing knowledge on renal allograft immunopathology. Due to technical reasons, this method has been only rarely applied in the past. The use of indirect immunohistochemistry for double-staining bears the risk of nonspecific cross reactions between the two staining sequences. To date, various procedures have been refined to avoid such cross reactions. Here we assessed the validity of three different protocols for indirect immunohistochemical double-staining on frozen sections of renal transplant biopsies ( n=12). Both colocalized antigens and antigens with a non-overlapping distribution were stained according to each of the three protocols. Differentiation between the two staining sequences was achieved by employing different colored substrates of alkaline phosphatase (protocol 1), different enzymes (peroxidase and alkaline phosphatase) together with the use of 3,3'-diaminobenzidine-tetrahydrochloride substrate in the first staining sequence (protocol 2), or primary antibodies from different species (protocol 3). Sensitivity and specificity of each protocol were determined by quantitative comparison with control single-stainings of adjacent sections. Sensitivity of the first staining sequence was about 100% with each of the three protocols investigated. In the second staining sequence, sensitivities of protocols 1 (50%) and 2 (54-66%) were much lower than of protocol 3 (100%). Specificity of the second staining sequence was only 44% with protocol 1 compared with 98% with protocol 2 and 100% with protocol 3. In conclusion, protocols 1 and 2 are not recommended for quantitative double-staining analyses. In contrast, protocol 3 provided maximum sensitivity and specificity, even for antigens that are colocalized on the same cell type. Thus, the use of primary antibodies from different species is by far the most reliable technique for quantitative double-staining analyses in renal allograft tissue.

Antigens, CD↗

PubMedDate not supplied

Comparison of immunohistochemistry and silver stain for the diagnosis of pediatric Helicobacter pylori infection in urease-negative gastric biopsies.

We compared immunohistochemical and silver stains of pediatric gastric biopsy sections for the identification of Helicobacter pylori infection with chronic inflammation and a negative urease screening test. Thirty-seven patients (age range 10 months to 21 years) whose gastric antral biopsies were negative for the rapid urease test (CLO(R)) but positive for lymphocytic infiltration were selected for a retrospective study. Specimens had been subjected to a rapid urease test (CLO(R)) and hematoxylin and eosin staining, and Dieterle silver staining and immunohistochemical staining specific for H. pylori were also performed. Twelve additional patients with urease-positive biopsies were used as controls. With Dieterle staining, 8/37 (22%) urease-negative biopsies contained organisms morphologically compatible with H. pylori, 21/37 (56%) contained organisms not compatible with H. pylori, and 8/37 (22%) were negative for organisms. Immunostaining confirmed 6/8 (75%) Dieterle-positive cases as being H. pylori, was negative in 2/8 (25%) Dieterle-positive cases, and was positive in 2/8 (25%) Dieterle-negative cases. Biopsies from 8/12 (67%) urease-positive specimens contained organisms seen with both Dieterle and immunohistochemical stains, and 4/12 (33%) were negative with both stains. Although both stains yielded comparable results with H. pylori-positive biopsies, Dieterle staining was potentially confusing because of nonspecific staining of other organisms. A significant proportion of (CLO(R))-negative biopsies was positive for H. pylori with special stains. We therefore recommend the use of immunohistochemical staining rather than silver staining in the evaluation of urease-negative gastric biopsies demonstrating chronic inflammation in children.

Adolescent↗

PubMed1988

The value and limitations of the Gram stain examination in the diagnosis of intraamniotic infection.

Gram stain examination of amniotic fluid is a method used for the rapid diagnosis of intraamniotic infection in patients with preterm premature rupture of membranes and preterm labor. The management of these patients relies heavily on the Gram stain results. Therefore, it is critical that the diagnostic value and limitations, optimal technique, and outcome correlates be precisely established. Most studies have focused on culture results rather than on Gram stain results to describe neonatal and maternal outcome. However, management is based on the Gram stain because culture results are not immediately available. One hundred eighty-seven amniocenteses were performed in 131 patients with preterm premature rupture of the membranes (n = 90) and preterm labor (n = 41). Spun and unspun Gram stains were performed. Centrifugation of the sample did not improve the sensitivity of the technique significantly. The agreement between the two methods was substantial (kappa index 0.89, p less than 0.001). The sensitivity of the Gram stain was 44.8% and the specificity was 97.6%. The sensitivity of the Gram stain was directly proportional to the number of bacteria present in amniotic fluid. In the presence of greater than 10(5) colony forming units per milliliter, 80% of the Gram stains were positive. The absence of both bacteria and white blood cells in a smear was associated with a negative culture of amniotic fluid in 95% of the cases. Clinical chorioamnionitis was associated with a positive Gram stain of amniotic fluid (p less than 0.001). There was a trend toward a higher incidence of endometritis in patients with a positive Gram stain compared with those with a negative Gram stain (p = 0.07). There was no neonatal infectious morbidity in patients with a true negative Gram stain. Patients with a false negative Gram stain had a 25% incidence of neonatal infectious complications (proved and suspected sepsis).

Adult↗

PubMed1989

Sudan stain of fecal fat: new insight into an old test.

The 72-h fecal fat determination is used as the gold standard to document the presence of steatorrhea. Although the Sudan stain for fecal fat is advocated as a sensitive screening test, a quantitative correlation between the 72-h fecal fat quantitation and the fecal Sudan stain is lacking. This study was designed to examine the staining properties of different classes of purified lipids in an experimentally defined artificial matrix, and to elucidate the reasons for the lack of quantitative correlation between these two tests. Our results indicate that the "neutral fat" stain without acidification or heating identifies triglyceride; and at an appropriate pH, the "neutral stain" also identifies fatty acid. The "split fat" stain with acidification and heating identifies both triglyceride and fatty acid. After acidification, fatty acid soaps are converted to the nonionized fatty acid. Thus, fatty acid soaps can be identified indirectly as fat droplets that are stained by the split fat stain. Although cholesterol is stained with Sudan stain after heating, upon cooling, cholesterol forms crystals of anhydrous cholesterol, making its staining pattern distinct. Neither the neutral fat nor the split fat stain can detect phospholipid or cholesteryl ester. The 72-h fecal fat determination is a measure of the total fatty acid content after a specimen is saponified. The resulting fatty acids are derived from a variety of endogenous and exogenous sources, including free fatty acids, soaps of fatty acids, triglycerides, cholesterol esters, and phospholipids. Therefore, the 72-h fecal fat quantitation does not differentiate between the primary sources of the measured fatty acid. It is concluded that the 72-h fecal fat determination is not specific for documenting triglyceride (fat) malabsorption. Until new methods are developed that specifically measure fecal triglyceride and fatty acid, the Sudan stain of fecal fat appears to be a more specific method for detecting the presence of triglyceride and fatty acid in a matrix.

Azo Compounds↗

PubMed2004

A high-affinity reversible protein stain for Western blots.

We describe a reversible staining technique, using MemCode, a reversible protein stain by which proteins can be visualized on nitrocellulose and polyvinylidine fluoride (PVDF) membranes without being permanently fixed to the membrane itself. This allows subsequent immunoblot analysis of the proteins to be performed. The procedure is applicable only to protein blots on nitrocellulose and PVDF membranes. MemCode is a reversible protein stain composed of copper as a part of an organic complex that interacts noncovalently with proteins. MemCode shows rapid protein staining, taking 30s to 1 min for completion. The method is simple and utilizes convenient application conditions that are compatible with the matrix materials and the protein. The stain is more sensitive than any previously described dye-based universal protein staining system. The turquoise-blue-stained protein bands do not fade with time and are easy to photograph compared to those stained with Ponceau S. Absorbance in the blue region of the spectrum offers good properties for photo documentation and avoids interference from common biological chromophores. The stain on the protein is easily reversible in 2 min for nitrocellulose membrane and in 10 min for PVDF membrane with MemCode stain eraser. The stain is compatible with general Western blot detection systems, and membrane treatment with MemCode stain does not interfere with conventional chemiluminescent or chromogenic detection using horseradish peroxide and alkaline phosphatase substrates. The stain is also compatible with N-terminal sequence analysis of proteins.

Blotting, Western↗

PubMed2004

Staining of proteins on SDS polyacrylamide gels and on nitrocellulose membranes by Alta, a colour used as a cosmetic.

We describe here the use of Alta, a pre-existing scarlet-red stain of cosmetic use, for staining proteins on sodium dodecyl sulfate (SDS) polyacrylamide gels, as well as for a single step staining of gels and nitrocellulose membranes during Western blot analysis. This stain, which is composed of 0.8% Crocein scarlet (brilliant crocein) and 0.2% Rhodamine B, is inexpensive, easy to use and nearly as sensitive as Coomassie Brilliant Blue (CBB) R-250. The gels can be stained in 10% Alta (2 h) and can be destained effectively only with 7% acetic acid as opposed to the conventional destainer (methanol/acetic acid/water) required for CBB-stained gels. In an alternative procedure, the proteins can be stained on the gel while electrophoresis by simply using 5% Alta in the top tank buffer and the stain can be viewed under UV-transilluminator. This procedure can also be used for Western blot analysis, as a single step procedure for staining of proteins on the gel as well as on the nitrocellulose membrane, as the stain is retained on the membrane after protein transfer. Thus, this staining procedure allows monitoring of proteins after each step in the Western blot, thereby eliminating the need to run separate gels for staining and Western blot analysis, and also the need for Ponceau Red S staining of the nitrocellulose membrane during Western blot analysis.

Azo Compounds↗

PubMedDate not supplied

Cryo-negative staining.

A procedure is presented for the preparation of thin layers of vitrified biological suspensions in the presence of ammonium molybdate, which we term cryo-negative staining. The direct blotting of sample plus stain solution on holey carbon supports produces thin aqueous films across the holes, which are routinely thinner than the aqueous film produced by conventional negative staining on a continuous carbon layer. Because of this, a higher than usual concentration of negative stain (ca. 16% rather than 2%) is required for cryo-negative staining in order to produce an optimal image contrast. The maintenance of the hydrated state, the absence of adsorption to a carbon film and associated sample flattening, together with reduced stain granularity, generates high contrast cryo-images of superior quality to conventional air-dry negative staining. Image features characteristic of unstained vitrified cryo-electron microscopic specimens are present, but with reverse contrast. Examples of cryo-negative staining of several particulate biological samples are shown, including bacteriophage T2, tobacco mosaic virus (TMV), bovine liver catalase crystals, tomato bushy stunt virus (TBSV), turnip yellow mosaic virus (TYMV), keyhole limpet hemocyanin (KLH) types 1 and 2, the 20S proteasome from moss and the E. coli chaperone GroEL. Densitometric quantitation of the mass-density of cryo-negatively stained bacteriophage T2 specimens before and after freeze-drying within the TEM indicates a water content of 30% in the vitreous specimen. Determination of the image resolution from cryo-negatively stained TMV rods and catalase crystals shows the presence of optical diffraction data to ca. 10 A and 11.5 A, respectively. For cryo-negatively stained vitrified catalase crystals, electron diffraction shows that atomic resolution is preserved (to better than 20 diffraction orders and less than 3 A). The electron diffraction resolution is reduced to ca. 10 A when catalase crystal specimens are prepared without freezing or when they are freeze-dried in the electron microscope. Thin vitrified films of TMV, TBSV and TYMV in the presence of 16% ammonium molybdate show a clear indication of two-dimensional (2-D) order, confirmed by single particle orientational analysis of TBSV and 2-D crystallographic analysis of TYMV. These observations are in accord with earlier claims that ammonium molybdate induces 2-D array and crystal formation from viruses and macromolecules during drying onto mica. Three-dimensional analysis of the TBSV sample using the tools of icosahedral reconstruction revealed that a significant fraction of the particles were distorted. A reconstruction from a subset of undistorted particles produced the characteristic T = 3 dimer clustered structure of TBSV, although the spikes are shortened relative to the structure defined by X-ray crystallography. The 20S proteasome, GroEL, catalase, bacteriophage T2, TMV, TBSV and TYMV all show no indication of sample instability during cryo-negative staining. However, detectable dissociation of the KLH2 oligomers in the presence of the high concentration of ammonium molybdate conforms with existing knowledge on the molybdate-induced dissociation of this molecule. This indicates that the possibility of sample-stain interaction in solution, prior to vitrification, must always be carefully assessed.

Animals↗

PubMed2003

Staining characteristics of preserved human amniotic membrane.

PURPOSE: Amniotic membrane is an ultra-thin cellophane-like membrane that is used in ocular surface reconstruction. We evaluated the staining characteristics of commonly available dyes on preserved human amniotic membrane to aid in handling of amniotic membrane during transplantation. METHODS: Five dyes, indocyanine green (2.5%, 1.0%, and 0.5%), fluorescein (0.25%), rose bengal (1%), lissamine green B (1%), and trypan blue (0.5%), were used to stain amniotic membrane. After staining, the specimens were observed under a dissecting microscope to evaluate for the uptake of the stains. Positively stained membranes were evaluated for the persistence of staining by placing them in 2 to 3 mL of balanced saline solution that was changed every 30 minutes over 6 hours. RESULTS: Preserved human amniotic membrane is stained by indocyanine green, rose bengal, lissamine green B, and trypan blue. Of these four dyes, only the membrane stained with 1% lissamine green B was free of stain after 120 minutes. Indocyanine green, rose bengal, and trypan blue continued to strongly stain the membrane after 24 hours. CONCLUSIONS: Indocyanine green, rose bengal, trypan blue, and lissamine green B all stain amniotic membrane. Lissamine green B appears to have advantages over the other dyes in that it will stain the membrane well, and in our model, dissipate in 120 minutes. Intraoperative staining with lissamine green B may be a simple and effective way to assist surgeons in the proper handling of amniotic membrane.

Amnion↗

PubMed1998

Day to day variations in ocular surface staining of the bulbar conjunctiva.

PURPOSE: The purpose of this study was to characterize the variability of bulbar conjunctival staining over time in response to a single application of fluorescein. METHODS: Sixteen subjects were stained Monday through Friday for a 2-week period. Appointments were scheduled from 9:00 a.m. to 12:00 p.m. to minimize variance due to diurnal variation. Fluorescein stain was applied by touching a separate Ful-Glo strip wetted with nonpreserved saline to the lower palpebral conjunctiva of each eye, with the right eye being stained first. Immediately after application of the fluorescein, conjunctival staining was graded in a randomized order by two examiners, using cobalt blue illumination and a no. 15 Wratten enhancement filter. Conjunctival staining was assessed based upon a 0 to 4 grading scale with 0.5 grade increments. By evaluating the variability in staining over time for each subject, a representation of overall fluctuation for the group was obtained. RESULTS: The average duration for an episode of staining graded higher than the group median (grade 0.5) was found to be 2.0 +/- 2.4 days. Overall, a mean staining grade of 0.5 for the right eye and 0.6 for the left eye was found. Right and left eyes were found to correlate well (r = 0.67, p < 0.01, Spearman's correlation) and the mean difference was not statistically significant [p = 0.73, multi-way, repeated measures analysis of variance (ANOVA)]. The mean difference in grading between the two examiners was 0.1 grades (p = 0.67, multi-way, repeated measures ANOVA), with a correlation coefficient of r = 0.68 (p < 0.01, Spearman's correlation). CONCLUSIONS: Characterization of conjunctival staining over time may be useful in evaluating clinically suspect staining, and in determining its potential duration. The appearance and variability of the conjunctival staining patterns noted in this study indicate a potential relationship to normal physiological processes, such as desquamation. This study also demonstrated a low amount of variability between right and left eyes, supporting the use of contralateral studies in the assessment of conjunctival staining. It also demonstrated a good consistency between separate examiners despite not being standardized.

Adult↗